1. Field of the Invention
The present invention relates to the provision of novel means for the diagnosis and therapy of cancers characterized by overexpression of gene products from the myc family. These means essentially include the use of monoclonal antibodies recognising an epitope common to acidic and basic isoferritins.
2. Description of the Related Art
Ferritin is a protein with a molecular weight of 440000 kDa used to store iron in cells. Since 1942, the date at which GRANICK isolated hepatic ferritin and until recently, this molecule was considered to be a uniquely cellular iron storage protein mainly located in the liver, the spleen and the bone marrow. It only appears in the serum in the event of a substantial overload of iron or during hepatic necrosis with liberation of this tissue protein into the vascular space. More sensitive detection methods using radioactive, enzymatic or fluorescent tracers have demonstrated the existence of a base concentration of serum ferritin and have allowed the study of variations thereof during the course of various diseases. At the same time, a number of research teams have endeavoured:                to refine knowledge regarding the biochemical structure of tissue ferritin;        to discover the reasons for the heterogeneity of the molecular forms grouped under the term “isoferritins”;        to compare the different characteristics of ferritin extracted from plasma or from different healthy or diseased organs.        
Current data show that ferritin is a macromolecular structure constituted by a glycoprotein structure in the form of a shell, Apoferritin, which is the protein component and which encloses a metallic core of iron atoms distributed in iron crystals inside its cavity.
X ray crystallographic analysis of ferritin has shown the presence of 24 sub-units, distributed in a rigorous symmetry. The sub-units are of two types, H and L.
The genes coding for each of the sub-units have been cloned and their chromosomal location is known (Murow H. N., Aziz N., Leibold E. A. et al., The ferritin gene's expression and regulation, Ann. NY Acad. Sci. 1988, 528 113). The H type sub unit, with a molecular weight of 21 000, is in the majority among the most acidic isoferritins such as ferritin extracted from HeLa cells or ferritin of cardiac origin (H=heart), but it is also present to a lesser extent in the liver and spleen. The second type of sub-unit (L), with a molecular weight of 16000, is more specifically in the majority in the most basic molecules of hepatic origin (L=liver) or originating in the spleen.
The iron stored in ferritin is mobilised for the synthesis of proteins that incorporate ferrous or ferric ions as functional components (haemoglobins and enzymes such as cytochromes and catalases). The principal functions of ferritin in the liver, spleen and marrow are firstly to protect tissues from oxidation and damage caused by free radicals produced by interactions with iron, water and oxygen, and secondly to re-use iron for the synthesis of haemoglobins. In other organs or in plasma, ferritin contains little or no iron and its function is poorly understood.
It has long been known that different ferritin phenotypes exist (isoferritins) (Drysdale J. W. (1977), Structure and metabolism, (Ciba Foundation Symposium 51, New Series), pages 41–57. In 1965, Richter demonstrated that ferritin prepared from human liver and carcinomatous epidermoid HeLa cells had different electrophoretic migration rates, that could not be explained by differences in the iron content. In total, about twenty isoferritins were characterized.
Techniques such as electrofocussing, ion exchange chromatography and two-dimensional electrophoresis under denaturing conditions showed that isoferritins are formed by variable combinations of the two sub-units H and L (Murow et al., supra).
The role of basic isoferritins is well known, particularly that of serum ferritin in phenomena directly linked to iron metabolism. That of acidic ferritins is controversial: many authors have used it as a tumoral marker in man. In 1968, a α2H globulin, later termed α2H isoferritins was discovered in serum from patients with different neoplasias (Buffe D. et al., Presence d'une protéine d'origine tissulaire α2-Hglobuline dans le serum de sujets atteints d'affections malignes [Presence of a protein originating from tissue α2H globulin in the serum of subjects with malignant disorders], Int. J. Cancer (1968) 3: 850–856). Marcus D et al., (J. Natl. Cancer Disti. (1975) 55: 791–795) described an abnormal ferritin in the serum from patients with breast cancer. Drysdale J. et al., (Cancer Res. (1974) 34: 3352–3361) described an acid isoferritin identical to the ferritin contained in HeLa cells with a particularly high concentration in different types of neoplasia. Moroz C. et al., (Clinical Exp. Immunol. (1977) 29: 30–76) describe a factor, which is an isoferritin, secreted by a sub-population of T lymphocytes present in particular in breast cancer and Hodgkin's disease. Finally, it should be noted that the majority of human neoplastic processes (Hodgkin's disease, breast, ovarian, pancreas, lung cancers, epidermoidal cancers of the head and neck, neuroblastoma, acute lymphoblastic leukaemia (ALL), Kaposi sarcomas, etc. . .) are accompanied by an elevation in serum ferritin. In all cases, a high level of serum ferritin is a negative prognostic factor (Hann H. W., Evans A. E., Siegel S. E. et al., Cancer Res., (1985) 45; 2843–2848; Jacobs A., Slater A., Wittaker J. A. et al., J. Cancer, (1976) 34: 533).
It has also been shown that in several types of cancer, there is a local tumoral increase in tissue ferritin, although the stromal or purely tumoral reactional origin of this excess ferritin has not been formally defined. Nevertheless, all authors are in agreement that it concerns acidic ferritin.
The development of antibodies and in particular monoclonal antibodies in imaging or for the treatment of certain diseases has been the aim of many developments.
A review of these developments was published in 1998 (P. S. Multani et al., (1998), Journal of Clinical Oncology, vol. 11. 16: 3691–3710). The diagnostic or therapeutic use of antibodies in a cell screening system with substances with a selective toxicity to specifically eliminate pathological cells is known as the magic bullet.
Briefly, the different possibilities for using monoclonal antibodies in this context are as follows:                a) the use of native antibodies as an immune effector, used in anti-tumoral therapies with a CDCC (complement-dependent cell cytotoxicity) activity or an ADCC (antibody-dependent cell cytotoxicity) activity. The antibody with a certain specificity as regards this target cell via its Fab fragments can then cause a cellular immune reaction due to Fc fragments of the same antibody. It can also involve blocking a receptor of the target cell or an anti-idiotypic vaccination specific for the tumoral antigen;        b) the antibody can be coupled to a toxin or a drug; the antibody is then a vector that transports said toxin or drug to the target;        c) antibodies can also act as a galenical vector by coupling with liposomes or other analogous systems into which cytoxic substances or drugs or anti-sense oligonucleotides, etc . . . can be introduced.        d) the antibodies can be directly or indirectly radiolabelled (chelate or bispecific antibody with an “antichelate, etc” site) and their radiation can be used for therapeutic ends or for imaging. A number of possible isotopes exist, in particular for labelling the antibodies. The most frequently used isotope is iodine 131. This method has a number of disadvantages (iodine fixing in the thyroid, existence of endogenous dehalogenases). Further, iodine 131 does not emit pure β radiation. Finally, its range length is short (1.1 mm).        
Coupling to indium 111 for imaging or yttrium 90 for therapy has also been developed. In that case, coupling is generally carried out by grafting a chelate to an antibody, the chelate fixing the radioactive emitter using techniques described and developed by the team headed by S. M. Quadri and H. M Vriesendorp (Vriesendorp H. M. et al., (1991), J. Clin. Oncol. 9: 918–928; P. E. Borchardt et al., (1998), The Journal of Nuclear Medicine 39: 476–484). The choice of yttrium 90 as a therapeutic radio-isotope is pertinent to cancerology. That isotope emits a pure β radiation; its half-life is 67 hours and its range length is 6.6 mm. It appears to be well adapted to treating solid tumoral masses, in particular Hodgkin's disease and malign non Hodgkin's lymphomas. Encouraging results have been obtained by the same team (H. M. Vriesendorp et al., (1995), Cancer Research 55, 58–92). These authors describe trials with polyclonal antiferritin antibodies labelled with Y 90 in patients with Hodgkin's disease resistant to conventional treatment methods combining chemotherapy and external radiotherapy and bone marrow grafts.
One of the biological characteristics of this disease is hyperexpression of ferritins by tumoral cells and the reactional cells surrounding them. Imaging tumour sites (indium 111) and the treatment of resistant forms of this disease by the system described in Vriesendorp et al., (1995), Cancer Research 55: 58–92 has produced good results.
Further, it is known that the myc oncogene family (the most important are c, N. L) codes for nuclear proteins that bind to DNA. In a normal cell, transcription of myc genes increases in the hours following a mitogenic signal. Proteins coded by the genes from the myc family have a “leucine zipper” moiety. This structure enables the formation of heterodimers (with c-fos or c-jun) or homodimers. Currently, the function of the c-myc product is viewed as being that of regulation of the transcription of cellular genes involved in mitosis initiation. In summary, myc genes acquire oncogenic properties by mechanisms that result in an overexpression of their products. This overexpression can be a result of:                gene amplification; or        activation of gene transcription; or        post-transcriptional modifications involving increased stability of mRNA. In vivo studies on the overexpression of the products of genes from the myc family in different tissues by the creation of transgenic animals has confirmed these facts. Finally, any increase in proliferation is physiologically accompanied by an increased expression of genes from the myc family.        
The three principal members of the myc family (c, N, L) are among the most frequently found oncogenes in human cancers that are activated by these three different mechanisms. In particular, c-myc is found in malignant lymphomas, L-myc and N-myc in small cell lung cancers and neuroblastomas from which the latter has been characterized. In the majority of malign human tumours, the degree of expression of myc genes is a negative prognostic factor.
Known target genes for which transcription is regulated by the products of genes from the myc family are few. The gene coding for the H chain of ferritin is the latest gene for which it has just been shown that it is negatively regulated by the product of the c-myc gene (Wu et al., Coordinated regulation of iron controlling genes, H ferritin and IRP2 c-myc, Science (1999) 283: 676–679). In other words, expression of the c-myc gene and/or other members of this family in cancer cells is inversely proportional to ferritin expression.